Nitrate and nitrite are anions occuring in a wide array of natural systems. In the environment both species are produced in the nitrification process, in which ammonia is oxidized by certain soil bacteria. Nitrate occurs in abundance in part because of the use of nitrate salts as fertilizers. Also, nitrite results through the reduction of nitrate by denitrification bacteria. This process is thought to be important in the occurrence of nitrite in the human intestine. Humans are exposed to nitrates and nitrites through the injection of vegetables, water and cured meats. Nitrite in high levels can be fatal to infants causing a condition known as methemoglobinemia.
It can therefore be seen that there is a continuing need for a selective analytical test system which determines nitrate and nitrite content. This is even more true as continuing biological and medical research shows increasing evidence of the physiological activity of these anions with respect to continued human exposure. Also, there is a continuing need for a sensitive and selective system of nitrate and nitrite analysis which can be conducted in the presence of turbid and colored fluids such as some biological fluids like urine and blood.
Then too, there is a need for an analytical technique which is specific to the nitrate-nitrite anions. That is to say, it determines only nitrate and/or nitrite without also determining total nitrogen content.
Yet another continuing need is for a system which can detect nitrate and nitrite even at the parts per billion level.
In the past most analytical techniques have not been able to differentiate between nitrate and nitrite and have simply determined total nitrogen content. Some techniques which have previously been used for nitrate and nitrite determination are the following. The most common method for nitrite involves the reaction of nitrite with sulfanilamide in acidic solution to yield a diazonium salt which is coupled with an aromatic amine to produce a highly colored azo compound. Methods for the spectrophotometric determination of nitrate are generally based on nitration of a phenolic type compounds, oxidation of an organic compound by nitrate, or reduction of nitrate to nitrite and determination via the sulfanilamide method. These methods have been reported to be subject to various interferences, thus requiring elaborate clean-up procedures when working with biological media. Ion chromatography has been applied to the determination of nitrate and nitrite in environmental samples. Although this method is more selective than those based on colorimetry, it is limited by a lack of sensitivity.
Both nitrate and nitrite have been determined volumetrically or manometrically by reduction to various gaseous species. An ultraviolet absorption method for nitrate by reduction to ammonia has been published. Methods have also been described in which nitrate and nitrite were determined by thermal reduction to nitric oxide followed by chemiluminescence detection. In general the chemical reduction of nitrate required highly acidic conditions except when vanadium (II) was used as a reducing agent. Although volumetric methods are applicable only to samples containing relative large amounts of nitrate or nitrite, under the appropriate kinetic conditions the reactions are presumed to be applicable to much lower amounts of nitrate or nitrite.
None of these systems have been sufficient to fulfill the needs earlier mentioned herein.
It is therefore a primary objective of the present invention to provide a technique of determination of nitrate-nitrite at extremely low levels, which is highly selective in that it determines only nitrate and nitrite without also determining total nitrogen content, which can be quickly, efficiently and easily run, which can be run on extremely small samples, which can be run on fluid samples of high turbidity and color such as urine and blood, and which, if run in a specific sequence, can differentiate between nitrite and nitrate. This is accomplished by first reducing and determining nitrite with a weak reducing agent under mild conditions and thereafter determining the nitrate under stronger reducing conditions.
The manner of fulfilling each of the needs, goals and objectives of this invention will be set forth hereinafter.